Despite many advances in treatment, diabetes continues to be the leading cause of chronic renal failure, adult blindness and limb amputation, and a major risk factor for heart disease, stroke and birth defects. To decrease the burden of this disease, a better understanding of the molecular mechanisms regulating insulin secretion by b-cell and insulin action in peripheral tissues is urgently needed, so that new interventions can be developed. Our strategy is to gain this knowledge through genetic studies of maturity-onset diabetes of the young (MODY) - an autosomal dominant form of early-onset diabetes. This approach has led during the previous funding period to the identification of the tyrosine kinase BLK as a previously unrecognized modulator of b-cell function that acts as a stimulator of insulin synthesis and secretion in response to glucose. In this grant renewal application, we propose to continue this research by seeking further insights onto the molecular mechanisms through which BLK modulates b-cell function and by pursuing the positional cloning of a new MODY gene placed on chromosome 4q32, where we have observed significant linkage in the Joslin collection of MODY families. Our specific aims are: 1. To investigate the molecular mechanisms by which the MODY gene BLK impacts b-cell function. We will perform in vitro and in vivo studies to systematically examine the effects of the BLK gene on b-cell function, proliferation, and survival. We will first investigate the effects of gain or loss of function of BLK in MIN6 b-cells. We will then study an existing global BLK KO mouse as well as an inducible-b-cell-specific BLK KO mouse that we will create during this grant. Mice will be phenotyped through detailed physiological studies to characterize their glucose homeostasis and b-cell function. 2. To identify and functionally characterize the sequence variants underlying the new MODY locus on chromosome 4q32. We will use next-gen sequencing technologies to compile a comprehensive catalog of the rare variants carried by the diabetes-linked haplotypes at 4q32. From these, we will select a set of candidate variants that have a high in silico likelihood of being involved in the etiology of MODY in these families. We will then determine which of these variants are causally linked to diabetes by means of functional studies. Several important features distinguish our proposal. First, it is based on extensive preliminary data concerning the role of BLK in b-cell physiology and demonstrating the feasibility of using next-gen sequencing for positional cloning. Second, it takes advantage of one of the largest available collections of MODY families unaccounted for by known MODY genes. Third, it builds on the ongoing collaboration between a genetic epidemiologist and a b-cell molecular physiologist with complementary expertise. Finally, and most importantly, our proposal has an especially high translational value, potentially leading to the identification of novel drug targets, with critical implications for the development of new interventions to curb the on-going diabetes epidemic. PUBLIC HEALTH RELEVANCE: The goal of this project is to identify genes involved in the development of MODY and understand the mechanisms through which these affect glucose homeostasis. Availability of new genetic markers of MODY would facilitate the diagnosis of this form of diabetes, with implications for treatment optimization (e.g., for transferring previously undiagnosed MODY from insulin therapy to oral medications). Perhaps more importantly, understanding the function of the genes may point to as yet undiscovered molecular pathways regulating insulin secretion and action, which may in turn suggest novel targets for the development of new drugs to prevent or treat common, multifactorial forms of type 2 diabetes.